The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia

After a dose of 10 micrograms of 6-aminonicotinamide (6-AN) was administered to day-4- chick embryo in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis. The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [35S]sulfate and [3H]glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the 35S/3H ratio, the proportion of unsulfated disaccharide (delta Di-0S), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of 35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilage-specific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation. We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder, i.e., micromelia.     

Requirement of ZO-1 for the formation of belt-like adherens junctions during epithelial cell polarization

The molecular mechanisms of how primordial adherens junctions (AJs) evolve into spatially separated belt-like AJs and tight junctions (TJs) during epithelial polarization are not well understood. Previously, we reported the establishment of ZO-1/ZO-2-deficient cultured epithelial cells (1[ko]/2[kd] cells), which lacked TJs completely. In the present study, we found that the formation of belt-like AJs was significantly delayed in 1(ko)/2(kd) cells during epithelial polarization. The activation of Rac1 upon primordial AJ formation is severely impaired in 1(ko)/2(kd) cells. Our data indicate that ZO-1 plays crucial roles not only in TJ formation, but also in the conversion from "fibroblastic" AJs to belt-like "polarized epithelial" AJs through Rac1 activation. Furthermore, to examine whether ZO-1 itself mediate belt-like AJ and TJ formation, respectively, we performed a mutational analysis of ZO-1. The requirement for ZO-1 differs between belt-like AJ and TJ formation. We propose that ZO-1 is directly involved in the establishment of two distinct junctional domains, belt-like AJs and TJs, during epithelial polarization.     

Characterization of the genus Bifidobacterium by automated ribotyping and 16S rRNA gene sequences

In order to characterize the genus Bifidobacterium, ribopatterns and approximately 500 bp (Escherichia coli positions 27 to 520) of 16S rRNA gene sequences of 28 type strains and 64 reference strains of the genus Bifidobacterium were determined. Ribopatterns obtained from Bifidobacterium strains were divided into nine clusters (clusters I-IX) with a similarity of 60%. Cluster V, containing 17 species, was further subdivided into 22 subclusters with a similarity of 90%. In the genus Bifidobacterium, four groups were shown according to Miyake et al.: (i) the Bifidobacterium longum infantis-longum-suis type group, (ii) the B. catenulatum-pseudocatenulatum group, (iii) the B. gallinarum-saeculare-pullorum group, and (iv) the B. coryneforme-indicum group, which showed higher than 97% similarity of the 16S rRNA gene sequences in each group. Using ribotyping analysis, unique ribopatterns were obtained from these species, and they could be separated by cluster analysis. Ribopatterns of six B. adolescentis strains were separated into different clusters, and also showed diversity in 16S rRNA gene sequences. B. adolescentis consisted of heterogeneous strains. The nine strains of B. pseudolongum subsp. pseudolongum were divided into five subclusters. Each type strain of B. pseudolongum subsp. pseudolongum and B. pseudolongum subsp. globosum and two intermediate groups, which were suggested by Yaeshima et al., consisted of individual clusters. B. animalis subsp. animalis and B. animalis subsp. lactis could not be separated by ribotyping using Eco RI. We conclude that ribotyping is able to provide another characteristic of Bifidobacterium strains in addition to 16S rRNA gene sequence phylogenetic analysis, and this information suggests that ribotyping analysis is a useful tool for the characterization of Bifidobacterium species in combination with other techniques for taxonomic characterization.     

67-kDa Laminin Receptor-dependent Protein Phosphatase 2A (PP2A) Activation Elicits Melanoma-specific Antitumor Activity Overcoming Drug Resistance

The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.     

FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas

The axoneme—the conserved core of eukaryotic cilia and flagella—contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo–electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beak''s localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra–B-tubular substructures required for a planar asymmetrical waveform.     

Xenogenic macrophage immunization reduces atherosclerosis in apolipoprotein E knockout mice

Atherosclerosis is a complex chronic inflammatory disease in which macrophages play a critical role, and the intervention of the inflammatory process in atherogenesis could be a therapeutic strategy. In this study, we investigated the efficacy of xenogenic macrophage immunization on the atherosclerotic lesion formation in a model of murine atherosclerosis. Apolipoprotein E knockout (apoE-KO) mice were repeatedly immunized with formaldehyde-fixed cultured human macrophages (phorbol ester-stimulated THP-1 cells), using human serum albumin as a control protein or HepG2 cells as human control cells, once a week for four consecutive weeks. The vehicle phosphate-buffered saline was injected in the nonimmunized controls. THP-1 immunization induced antibodies that are immunoreactive with mouse macrophages. Although the plasma lipid levels were unchanged by the immunization, the atherosclerotic lesion area in the aortic root was significantly reduced by >50% in 16-wk-old THP-1-immunized apoE-KO mice compared with that in control mice. THP-1 immunization reduced in vivo macrophage infiltration, reduced in vitro macrophage adhesion, and changed cytokine production by macrophages to the antiatherogenic phenotype. Xenogenic macrophage immunization protects against the development of atherosclerosis in apoE-KO mice by modulating macrophage function in which antibodies induced by the immunization are likely to be involved. This method is a novel and potentially useful cell-mediated immune therapeutic technique against atherosclerosis. antibody; THP-1 cells     

Phylogeny and biogeography of the Anderson's crocodile newt, Echinotriton andersoni (Amphibia: Caudata), as revealed by mitochondrial DNA sequences

The Anderson's crocodile newt, Echinotriton andersoni, is considered a relic and endangered species distributed in the Central Ryukyus. To elucidate phylogenetic relationships and detailed genetic structures among populations, we analyzed variation in the mitochondrial cytochrome b gene. Results strongly support a primary dichotomy between populations from the Amami and Okinawa Island Groups with substantial genetic divergence, favoring a primary divergence between the two island groups. Within the latter, populations from the southern part of Okinawajima Island are shown to be more closely related to those from Tokashikijima Island than to those from the northern and central parts of Okinawajima. The prominent genetic divergence between the two island groups of the Central Ryukyus seems to have initiated in the Miocene, i.e., prior to formation of the strait that has consistently separated these island groups since the Pleistocene. The ancestor of the southern Okinawajima-Tokashikijima is estimated to have migrated from the northern and central parts of Okinawajima into southern Okinawajima at the Pleistocene, and dispersed into Tokashikijima subsequently.     

Isolation and characterization of a rice cDNA clone encoding ATP/ADP translocator

We isolated a rice cDNA clone which encodes an open reading frame of 382 amino acids. Its deduced amino acid sequence corresponds to an ATP/ADP translocator protein. Its homology with a maize ATP/ADP translocator was 83.9% in nucleotide sequence, and 90.2% of the amino acid level. Expression of this gene is regulated by such external stresses as salinity and low temperature.     

Effects of CD80 and CD86 on cytokine production in patients with wasp-venom allergy who receive venom immunotherapy

Several studies have provided evidence that activation of antigen-specific T cells requires interactions between CD28 on T cells and its ligands, CD80 and CD86, on antigen-presenting cells (APCs). However, the effects of CD80 and CD86 on cytokine production in patients with Hymenoptera venom allergy who receive venom immunotherapy remain unclear. We examined the effects of CD80 and CD86 on Th1- and Th2-cytokine production before and after venom immunotherapy in patients with wasp-venom allergy. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with wasp-venom allergy before and after three months of venom immunotherapy. CD4+ T cells and monocytes were isolated as APCs from PBMCs and were cocultured with wasp venom in the presence of anti-CD80 or -CD86 blocking antibodies. Interleukin (IL)-4, IL-10, and interferon (IFN)-gamma were measured by enzyme-linked immunosorbent assay. The expression of CD80 and CD86 on CD14+ PBMCs was detected by fluorescence-activated cell-sorter analysis. The expression of CD86, but not that of CD80, on CD14+ PBMCs cocultured with venom increased after three months of venom immunotherapy, but not before venom immunotherapy. Blockade of CD86 reduced IL-10 production after three months of venom immunotherapy. IL-10 production promoted by CD86 costimulation may be involved in the mechanism of venom immunotherapy in patients with venom allergy.